Ligand/anti-ligand assays for adherent proteins

ABSTRACT

The present invention provides ligand/anti-ligand assays for detecting and/or measuring adherent proteins, including lipophilic serum or plasma proteins, such as serum amyloid A (SAA) and apolipoprotein A1 (apoA1); cytokines such as IL-1 beta, IL-6, and TNF alpha; pentraxins, such as CRP; and globular serum or plasma proteins such as albumin.

This is a continuation-in-part of copending application Ser. No.07/421,205 filed on Oct. 13, 1989 now abandoned.

BACKGROUND OF THE INVENTION

The present invention relates to ligand/anti-ligand assays for detectionand measurement of adherent proteins, including lipophilic serum andplasma proteins, cytokines, globular serum and plasma proteins, andpentraxins. Assays of the present invention are particularly useful fordetection and measurement of serum amyloid A, apolipoprotein A1,apolipoprotein B, CRP, IL-1 beta, TNF alpha, albumin and similaradherent proteins.

Biological fluids such as plasma and serum contain numerous proteins.The presence, absence or concentration of a particular protein may be ofinterest because, e.g., such data provided information regarding theclinical state of the individual from which the biological fluid wasobtained. Accordingly, it is desired to have relatively simple andinexpensive assays to detect the presence of and determine theconcentration of such proteins.

By way of example, plasma and serum contain several classes of proteinswhich are non-convalently linked to lipids. These lipophilic proteinsperform a variety of functions, including lipid transport, intercellularcommunication, and host defense. Medical research has found that in anumber of disease states, levels of lipophilic serum and plasma proteinsdeviate from those found in non-disease states. The significance ofthese deviations is the subject of active clinical research.

The concentrations of many of the lipophilic serum or plasma proteinschange as the physical state of the body changes. For example, the levelof serum amyloid A dramatically increases during inflammation; the levelof apoliprotein A1 decreases during coronary disease, while that ofapolipoprotein B is elevated during coronary disease. Clinicalmeasurement of these kinds of proteins will therefore become moreimportant as more is learned about the mechanisms of various diseasestates.

Accurate and reproducible measurement of lipophilic serum or plasmaproteins has historically been difficult. Many factors contribute tothese difficulties, such as the existence of multiple forms of thelipophilic proteins and the inherent "stickiness" of the lipophilicproteins.

Organisms frequently contain several molecular forms of a particularlipophilic protein which differ only slightly in amino acid sequence.These molecular forms are known as isotypes, each of which may in turnmay exist in multiple conformational states. Immunological reactivitymay vary among isotypes and conformational states of a particularisotype.

Conformational changes are frequently observed in apolipoproteins whichaccompany the binding of lipid by the apolipoprotein. (Bausserman etal., J. Biol. Chem. 258, 10681 (1983); Segrest et al., Biochemistry 15,3187 (1976); Segrest et al., FEBS Letters 38, 247 (1974); Morrisett etal., Biochemistry 12, 1290 (1973)). It is possible to observe theseconformational changes when the protein in serum or plasma is analyzedby immunodiffusion or charge shift immunoelectrophoresis (Linke,Biochim. Biophys. Acta 668, 388 (1981)). Treatment with heating, acid,alkali, guanidine hydrochloride and extraction with organic solvents canalso induce changes in apolipoprotein conformation. (Sipe et al., Br. J.Exp. Path. 57, 582 (1976); Pepys and Baltz, Adv. Immunol. 34, 141(1983); Eriksen and Benditt, Meth. Enzymol. 128, 311 (1986); Maciejko,Clin. Chem. 28, 199 (1982)).

Moreover, lipophilic proteins are "sticky", i.e., they form non-specifichydrophobic interactions with other molecules of the same structure(also referred to as self association). This stickness also causesnon-specific hydrophobic interactions between the lipophilic proteinsand unrelated serum proteins and laboratory vessels. (Franklin, J. Exp.Med. 144, 1679 (1976); Marhaug and Husby, Clin. Exp. Immunol. 45, 97(1981); Bausserman et al., op. cit., (1983)). The inherent stickiness oflipophilic proteins causes inaccurate measurements of the proteins inimmunoassays.

Three representative lipophilic proteins present in serum and plasma areapolipoprotein A1, apolipoprotein B, and serum amyloid A. ApolipoproteinA1 (hereinafter apoA1) is one of the major proteins present inhigh-density lipoproteins (hereinafter HDL). Apo A1 may be a necessarystructural component of HDL (H. K. Naito, J. Clin. Immunoassay 9, 11(1986), and it is an activator of lecithin cholesterol acetyltransferase, an enzyme in the pathway which removes cholesterol fromperipheral blood. Apolipoprotein B (hereinafter apoB), the principalprotein constituent of low density lipoproteins (hereinafter LDL), isactive in recognition of cellular receptors for catabolism of LDL. Serumamyloid A (hereinafter SAA) is also associated with HDL, but thislipoprotein plays no known role in lipid transport. SAA is one of theacute phase reactants, i.e., it is present at elevated levels duringacute inflammatory states.

ApoA1 consists of a single unglycosylated chain of 243 to 245 amino acidresidues, which do not include cystine, cysteine, or leucine. Severalisotypes of ApoA1 exist, and the lipid-free state of the protein has analpha helical content of 55% which increases to 75% when phospholipid isbound to the apoprotein. ApoA1 is synthesized in liver and intestine.

The clinical importance of ApoA1 measurements lies in its utility inassessing coronary artery disease. As stated above, ApoA1 levels aredecreased in individuals with coronary disease and are therefore ofclinical significance. ApoB levels, in contrast, are elevated incoronary disease, and comparative measurement of ApoA1 and ApoB levelsprovides a sensitive clinical profile.

The concentration of SAA in plasma and other biological specimens isalso of clinical significance (Rosenthal and Franklin, J. Clin. Invest.55, 746 (1975); Gorevic et al., Clin. Immunol. Immunopathol. 6, 83(1976); Pepys & Baltz, op. cit., (1983); Sipe, in Laboratory DiagnosticProcedures in the Rheumatic Diseases, A. S. Cohen, ed., Grune andStratton, Orlando (1985), p. 77; Kushner & Mackiewicz, Disease Markers5, 1 (1986)). There is minimal SAA synthesis during homeostasis, butwithin a few hours after injury, two major and several minor isoforms ofSAA can be detected in plasma high density lipoproteins (hereinafterHDL) (Benditt and Eriksen, Proc. Natl. Acad. Sci. USA 74, 4925 (1977);Bausserman et al., J. Exp. Med. 152, 641 (1980); Benditt et al., Meth.Enzymol. 163, 510 (1988); Strachan et al., J. Biol. Chem. 264: 18368(1989)). The amount and duration of SAA production during the acutephase response to tissue injury and cell necrosis depend upon the typeof injury and its magnitude (McAdam et al., J. Clin. Invest. 61, 390(1978); Sipe, in Rheumatology and Immunology, A. S. Cohen and J. C.Bennett, eds., Grune and Stratton, Orlando (1986), p. 97).

Synthesis of SAA is regulated by secretory products of the macrophagesuch as interleukin-1, tumor necrosis factor, and interleukin-6 (Vogeland Sipe, Surv. Immunol. Res. 1, 235 (1982); Ganapathi et al., Biochem.Biophys. Res. Commun. 157, 271 (1988)). SAA is cleared and/or consumedfrom plasma more rapidly than most glycosylated acute phase proteins (L.L. Bausserman, in Amyloidosis, J. Marrink and M. H. vanRijswijk, eds.,Martinus Nijhoff, Amsterdam (1986), p. 337). SAA concentration is thus auseful indicator of the recent production and action of Il-1 and relatedcytokines.

In the past two decades, SAA has been studied as the precursor ofamyloid fibrils, as an apoprotein constituent of HDL, and, mostextensively, as an acute phase protein. Because the concentration ofcirculating SAA is a sensitive, specific and quantitative marker ofrecent tissue damage and cell necrosis, it is of interest to monitor SAAvalues in clinical practice. However, despite its potential usefulness,reliable clinical measurement of SAA and many other lipophilic proteinshas not been possible. This is in large part due to the physicochemicalproperties of these proteins.

There are two amphipathic helical regions in SAA, one in the aminoterminal portion of the molecule spanning residues 1-24, and the secondfrom residues 50 to 74 (Parmelee et al., Biochemistry 21, 3298 (1982)).Dramatic changes in conformation and solubility occur when the carboxylportion of some isoforms of the SAA molecule is removed by proteolyticcleavage to form amyloid A (AA). AA protein forms insoluble fibrilshaving the cross beta pleated sheet conformation and accumulating in theextracellular spaces of tissues (Benditt and Eriksen, J. Pathol. 65, 231(1971); Glenner, N. Eng. J. Med. 302, 1283 (1980)). The isolated fibrilsare reported to be minimally antigenic and immunogenic (Ram et al., Int.Arch. Allergy 34, 269 (1968)).

Although the major portion of SAA in plasma may be isolated with HDLproteins after hours of centrifugation in the presence of highconcentrations of salt, it has been reported that a portion of plasmaSAA is present in the nonlipoprotein fraction of plasma (Marhaug, etal., Clin. Exp. Immunol. 50, 382 (1982), Bausserman, personalcommunication). Because it is not bound to lipids, the SAA in thenonlipoprotein plasma fraction can be expected to have a differentconformation from that isolated with HDL proteins. A plasma sample couldthus conceivably contain both SAA conformers, i.e., free SAA andHDL-associated SAA. Conformational differences between the conformerscan affect the accuracy of immunological assays, since a particularantibody or antiserum may not recognize all of the epitopes exposed inthe various conformers. Similar phenomena may also occur because of theexistence of more than one isotype of SAA present in the sameindividual.

The technical difficulties surrounding accurate, reproduciblequantification of SAA concentration in plasma and other biologicalfluids have been widely noted (Marhaug, Scand. J. Immunol. 18, 329(1983); Pepys and Baltz, op. cit., (1983); Godenir et al., J. Immunol.Methods 83, 217 (1985); Benditt et al., op. cit., (1988)). It has oftenbeen reported that optimal immunochemical measurement requiresdenaturation and dissociation of SAA from lipids and apolipoproteins.Some laboratories have found that denaturation by heat, acid or alkaliincrease immunoreactivity and reproducibility of SAA measurements (Sipeet al., op. cit., (1976); van Rijswijk, Amyloidosis, Ph.D. Thesis,University of Gronigen, The Netherlands (1981); Eriksen and Benditt, op.cit., (1986). On the other hand, other investigators report that the useof denaturing treatments results in less satisfactory quantification(Benson and Cohen, Arthritis Rheum. 22, 36 (1979); Chambers and Whicher,J. Immunol. Methods 59, 95 (1983); Marhaug, op. cit. (1983)). The basisfor these differing observations is thought to lie in the epitopespecificity of the antibodies employed and in the particular type ofimmunoassay employed (direct or competitive binding radioimmunoassays(RIA) or enzyme-linked immunosorbent assays (ELISA) or radialimmunodiffusion).

Purified apoSAA may be measured by any of the traditional methods formeasuring proteins. However, methods such as gel scanning, amino acidanalysis, or high performance liquid chromatography are expensive andlabor intensive and are thus unsuitable for clinical use. Moreover,these methods require purification since serum and plasma containsubstances which may interfere with traditional protein assays.Furthermore, measurement of purified SAA may not accurately reflect thelevels of the native lipoprotein.

Immunoassays that measure SAA in the native state are highly desirable,since clinical laboratories routinely use these kinds of procedures, andsuch assays from the basis of a large portion of general clinicalliterature on many biological ligands. Several immunoassays for SAA havebeen described (DeBeer et al., 1982, Bausserman et al., 1988, Benson andCohen, 1979). However, the known assays of SAA are subject tointerference from other plasma constituents and have not provenclinically useful.

Moreover, the known clinically applicable immunoassays are onlysemi-quantitative, with sensitivities adequate for the high SAA levelsobserved in acute states such as pneumonia or trauma but inadequate formonitoring SAA in chronic patients. SAA levels in chronic rheumaticpatients can be as low as 5-100 μ/ml, and changes in SAA levels of achronic rheumatic patient as small as 10 to 20 μ/ml can be clinicallyrelevant. Monitoring of chronic patients necessitates, therefore, a moresensitive assay than has previously been available.

Finally, inflammatory stimulation stimulates circulating SAA levels byseveral hundred to a thousandfold. In order to be clinically useful,therefore, it is highly desirable that an assay for SAA be accurate overa concentration range of several orders of magnitude.

Marhaug (op. cit., 1983) compared a sandwich ELISA using polyclonalrabbit anti-AA and SAA and monoclonal mouse anti-SAA antibodies with aninhibition ELISA in which plates were coated with apoSAA, and testsamples were incubated with monoclonal anti-SAA antibodies prior toaddition to the wells. Both methods were affected by self-coating withSAA. Denaturation was not required and delipidation resulted in slightlyreduced immunoreactivity. Neither assay provided the sensitivityrequired for clinically relevant measurements of SAA. Marhaug alsodescribed a radioimmunoassay, which he found to be more accurate thaneither ELISA. However, radioimmunoassay has numerous drawbacks inherentto use of radioactivity, and is thus not the method of choice forclinical measurement of SAA.

Dubois and Malmendier (J. Immunol. Methods 112, 71 (1988)) describedouble sandwich ELISA methodology for measurement of humanapolipoprotein S (probably identical to SAA) that utilizes peroxidaseconjugated anti-SAA antibodies to quantify apoS bound to wells coatedwith affinity purified anti-apoSAA. The sensitivity of this assay wasadequate. However, the assay is very labor intensive because of therequirement for affinity purification of antibodies and conjugates.Moreover, the expense of the reagents is higher than is desirable forroutine clinical use.

Zuckerman and Surprenant (J. Immunol. Methods 92, 37 (1986)) described amethod in which SAA was directly coated from mouse serum at 4° C. inbicarbonate buffer pH 9.6. Like most of the previous assays, this assayexpresses SAA concentrations in relative units rather than absoluteamounts. As indicated above, expression of SAA concentrations inrelative units limits comparison of values from samples obtained andassayed over a period of time and limits comparison of results amonglaboratories. Without such juxtaposition the clinical utility of any SAAassay is severely limited.

The methodology of Zuckerman and Suprenant is relatively quantitativefor mouse SAA when samples were assayed at the same dilution. Thismethod can be modified for measurement of SAA in human samples by usingSAA-rich lipoprotein fractions to construct standard curves which wouldbe more stable than those obtained with plasma. Samples within a groupmay then be assayed at the same dilution and the SAA concentrationexpressed in relative amounts of SAA-rich HDL.

However, the Zuckerman and Suprenant method has not proven suitable forclinical measurement of SAA in human plasma samples. In order to obtainmeaningful comparisons, clinical samples must be assayed over a SAAconcentration range of several orders of magnitude. This necessitatesassay of multiple sample dilutions of varying protein concentrations.Human plasma samples contain interfering proteins which affect bindingin different ways at different sample dilutions. It is postulated thathuman SAA may interact more strongly with other plasma constituents thandoes mouse SAA. The method of Zuckerman and Surprenant contains nocorrective provision for the variable interferences found in humanplasma samples.

Moreover, SAA from human plasma has been found to bind less efficientlyto microtiter plates than does SAA from mouse plasma or serum under theZuckerman and Surprenant conditions. Another complication arises becausethe samples measured by Zuckerman and Surprenant were from mice whichhad experienced experimental inflammatory stimulation. Consequently, SAAlevels in the mice were high as compared with the relatively low SAAconcentrations associated with human disease. It is believed that thesensitivity of the modified Zuckerman and Suprenant assay is thereforenot sufficient to measure SAA levels in human clinical samples.

Antigen capture systems using a double sandwich solid phase ELISA withpolyclonal rabbit anti-human SAA antiserum as the detection antibodyhave also proven ineffective for measurement of SAA in serum and plasmasamples. The double sandwich solid phase ELISA yielded high resultswhich were also variable. See, e.g., Marhaug (op. cit. 1983.) Thismethod immobilizes antibody on casein-blocked microtiter plates and theanalyte is added in solution. Moreover, the same amount of SAA bound tothe casein-blocked plates whether or not antibody was immobilized on theplates. When some of the same samples were purified and assayed by themodified Zuckerman/Surprenant method, the SAA levels were observed to bemuch lower than the values from the double sandwich solid phase ELISA.These results suggest that the double sandwich solid phase ELISA for SAAwas not accurate because of interactions between the SAA in the samplewith the blocking agent, among SAA molecules in the sample, or betweenSAA molecules and other serum constituents such as albumin, fibronectin,or SAP.

Covalent binding of SAA to Co-Bind plates (Micro Membranes, 95 OrangeStreet, Newark, N.J. 01720) has also been ineffective. Purified SAAbinds efficiently to the Co-Bind plates, showing greater levels ofbinding at pH 9.6 than at pH 7.2, which suggests that the increase in pHinduces conformational changes exposing free amino groups which bound tothe plates. Binding of purified SAA to the plates could be blocked,however, nonspecific binding was dramatically increased in plasmasamples. Denaturation by heat or guanidine treatment of samples prior tocapture was required to maximally expose determinants.

Direct binding of SAA from plasma to polyvinylchloride plates followedby delipidation with organic solvents is not sufficiently sensitive. Theobservation of Serban (U.S. Pat. No. 4,782,014) that SAA preferentiallybinds to plastic surfaces in the presence of a large excess ofirrelevant protein is of limited utility in perfecting an immunoassayfor SAA. Binding of SAA to the plastic surface must be controlledreproducibly and in such a way that a quantity directly proportional tothe concentration of SAA in the test sample is bound. Such controlledand reproducible binding was not present in Serban.

Benditt et al. (op. cit., 1988) describe a competitive inhibition ELISAmethodology in which plates are precoated with purified AA protein andthe heat denatured samples are incubated with affinity purified apo-SAAantibodies. Subtractive competition assays measuring the ability ofplasma samples to compete with antibody for binding to SAA antigencoated on plates was not sufficiently sensitive to detect SAA in the lowconcentrations present in clinical samples. Moreover, large quantitiesof antigen and antibody are required for this assay.

Other proteins found in biological fluids have been similarly difficultto assay. IL-1 beta and CRP are examples of such proteins.

Although it has been relatively easy to measure IL-1 beta in culturesupernatants by immunoassay, it has been difficult in plasma.Interference by plasma lipids and/or lipoproteins is indicated by Duff'slaboratory (Eastgate, J. A., et al., Lancet, p. 706, Sep. 24, 1988) inwhich chloroform extraction is performed prior to measurement of IL-1beta in the plasma of rheumatoid arthritis patients by ELISA.Accordingly, the present invention provides an improved assay for IL-1beta.

The two clinical methods most frequently used for CRP are nephelometryand radial immunodiffusion. Both methods have a threshold of 5-8 μ/ml.The methodology of the present invention provides for measurement of CRPin the range of 1-10 μ/ml which may be clinically useful for rheumatoidarthritis patients.

In order to be useful for quantitative clinical measurements, it ishighly desirable that an assay for any protein have the followingcharacteristics:

1. Potential to measure the ligand in essentially absolute amountsrather than relative units.

2. Potential for accurate measurement of the ligand over a clinicallyrelevant concentration range of the ligand;

3. Potential for automation of the assay.

4. Simple, reliable, inexpensive, and nonhazardous.

The ability to measure absolute units permits comparison of results fromsamples obtained and/or measured over a period of time and comparison ofresults from different laboratories. It is also desirable that the testbe capable of performance by multiple laboratory workers of relativelyunsophisticated skill levels.

Thus, improved methods for measuring levels of proteins, especiallythose technically difficult to measure, are being sought because of thedeficiencies of present methodologies.

SUMMARY OF THE INVENTION

Accurate and reproducible measurement of proteins having hydrophobicdomains is achievable through the practice of the present invention.While not wishing to be bound by theory, it is thought that thehydrophobic domains of such proteins form non-specific hydrophobicinteractions with various support media under the conditions of themethods of the present invention.

Accordingly, proteins which can be assayed in accordance with thepresent invention include those proteins which are capable ofnon-specific hydrophobic interactions with a desired support medium,particularly wherein such non-specific hydrophobic interactions arepromoted in the presence of salt at an elevated pH and temperature. Suchproteins are referred to herein as "adherent proteins." Accordingly, themethods of the present invention can be used to measure adherentproteins present in biological samples.

In accordance with the present invention, there is provided a method ofdetermining the presence or amount of an adherent protein of interest ina sample which comprises:

a. contacting the sample at high salt concentration and elevatedtemperature with a support medium having an affinity for the adherentprotein under conditions to promote binding of the adherent protein tothe solid support;

b. contacting the support medium obtained in step (a) with at least oneanti-ligand for the adherent protein under conditions to promote bindingof the anti-ligand to the adherent protein;

c. detecting and measuring the amount of anti-ligand bound to theadherent protein;

d. either (i) relating the amount of anti-ligand determined in step (c)with the amount of anti-ligand measured for at least one control sampleprepared in accordance with steps (a)-(c), said control samplecomprising one or more unrelated proteins known to be free of theadherent protein, or (ii) relating the amount of anti-ligand measured instep (c) with the amount of anti-ligand measured for samples containingknown amounts of adherent protein, said samples comprising purifiedadherent protein in a solution of one or more unrelated proteins andprepared in accordance with steps (a)-(c).

In accordance with the present invention, the salt concentration forbinding an adherent protein of interest to a support medium may varyfrom about 0.15M to approximate saturation for the particular saltchosen; the pH may vary from about 8 to about 11; and the temperaturemay vary from about 40° C. to 65° C. The support medium may be solid orliquid. The anti-ligand may be any molecule which binds specifically tothe adherent protein of interest, and any suitable method for detectionof binding may be used.

Unrelated proteins are preferably proteins which do not cross-reactimmunologically with the adherent protein of interest. Such unrelatedproteins may also aid in the binding of the adherent protein to asupport medium in a manner similar to the manner in which the adherentprotein would bind to the support medium if the biological fluid inwhich it is found were contacted therewith.

The application of this method to the measurement of serum amyloid A,apolipoprotein Al, apoliprotein B, CRP, IFN-1 beta, TNF-alpha andalbumin, respectively, is described in detail. The method is alsoapplicable to other adherent proteins, particularly those which aresimilarly difficult to measure.

It is expected that the methods of the present invention will be usefulin the assay of most proteins having one or more hydrophobic domains.The suitability of the methods of the present invention for assaying aparticular protein, can be determined, e.g., by preparing controlsamples containing the protein of interest in accordance with theteachings herein and, then, determining as taught herein whether theprotein in the control sample binds to the desired support medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, 1B, 1C, and 1D show densitometric analysis ofSDS-polyacrylamide (SDS is sodium dodecyl sulfate) gels of SAA purifiedas described in the Detailed Description of the Invention.

FIG. 2 shows the effect of temperature on SAA binding to microtiterplates.

FIG. 3A and 3B show the inhibitory effect of albumin on SAA binding towells of microtiter plates and its reversal by heating in the presenceof salt.

FIG. 4 depicts SAA standard curves prepared as described in the DetailedDescription of the Invention.

FIG. 5 is a comparison of the SAA ELISA of the present invention and adouble antibody RIA.

FIG. 6 is a representative standard curve for the ApoAl ELISA of thepresent invention, prepared as described in the Detailed Description ofthe Invention.

FIG. 7 is a comparison between the ApoA1 ELISA of the present inventionand a conventional immunoturbidimetric assay.

FIG. 8 shows a standard curve for an ApoB ELISA of the presentinvention.

FIG. 9 shows standard curves prepared in accordance with the presentinvention for IL-1 beta in the presence and absence of IgG/albumin.

FIG. 10 shows a standard curve prepared in accordance with the presentinvention for TNF/alpha.

FIG. 11 shows standard curves obtained in accordance with the presentinvention for purified human CRP in the presence (FIG. 11A) and absence(FIG. 11B) of purified human IgG and albumin.

FIG. 12 shows standard curves obtained in accordance with the presentinvention for purified serum albumin in the presence (FIG. 12A) andabsence (FIG. 12B) of purified human IgG.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the present invention are widely useful in determiningthe presence and concentration of adherent proteins in biologicalfluids, including lipophilic serum and plasma proteins, cytokines,globular serum and plasma proteins, and pentraxins. Such proteinsinclude SAA, ApoAl, ApoB, GRP, IL-1 beta, TNF alpha, albumin and otherproteins having a similar adherent nature.

The methods of the present invention are particularly useful in themeasurement of the adherent lipophilic proteins found in HDL, LDL, VLDLand associated with other plasma proteins.

Because the accurate measurement of lipophilic proteins has beenhistorically technically difficult, the methods of the present inventionwill be described in connection with these lipophilic proteins, as wellas other types of protein to show the wide usefulness of the subjectmethods.

Lipophilic proteins measured in accordance with the present inventionare preferably those which are difficult to measure accurately becauseof their conformational changes and isotypic degeneracies. The method ofthe present invention is most preferably used to measure lipophilicproteins such as SAA, ApoAl, ApoB, tumor necrosis factor, interleukin-6,interleukin-1 beta, and similar proteins found in association with HDL,LDL, VLDL and other plasma proteins and which vary measurably duringdisease, trauma, or genetically pathological states. Other adherentproteins which can advantageously be measured by the present inventioninclude CRP and albumin.

The methods of the present invention are preferably used to measureadherent proteins in large numbers of clinical samples which have beencollected at varying times and by varying personnel. The presentinvention is most preferably used to provide juxtaposable data from morethan one laboratory in large clinical studies.

In one preferred embodiment of the present invention, the methodcomprises contacting the sample to be measured with a support medium athigh salt and high pH for a time sufficient for the adherent protein ofinterest to bind to the medium, under conditions that promote binding ofthe adherent protein to the support medium. The support medium ispreferably a well or wells of a microtiter plate, a plastic bead, achromatographic resin, a magnetized bead, or other suitable supportwhich has affinity for the protein being measured. The support mediumcan also be a liquid medium in which more than one phase is present, ora liquid medium containing micellar structures. High salt concentrationin accordance with the present invention is any salt concentrationgreater than physiological salt concentration, i.e., greater than about0.15M. High salt concentration in accordance with the present inventionincludes salt concentrations approaching saturation of the particularsalt used. High pH in accordance with the present invention is any pHgreater than about 8. High pH in accordance with the present inventionincludes pH of about 11. The conditions which promote binding betweenthe adherent protein and the support medium can include varying thetemperature, varying the concentration of the adherent protein, additionof other solutes, or modifying other parameters specific to particularadherent proteins.

After the adherent protein is bound to the support medium, ananti-ligand specific for the adherent protein is contacted with theadherent protein bound to the support medium. Any anti-ligand to theadherent protein may be used, as long as the anti-ligand is specific forthe adherent protein. The anti-ligand is preferably an immunoglobulin, areceptor protein which binds specifically to the adherent protein, amembrane transport protein which binds specifically to the adherentprotein, or similar molecule. The immunoglobulin is preferably apolyclonal antiserum from any appropriately inoculated animal or amonoclonal antibody specific for the adherent protein. A simple orcomplex carbohydrate which binds specifically to the adherent proteincan also be used as an anti-ligand. In the case of lipophilic proteins,simple or complex lipids which bind specifically to the lipophilicprotein can be used.

The amount of anti-ligand bound to the adherent protein affixed to thesupport medium is then determined using any suitable detection methods.Any detection method can be used, such as conjugated antibodies to theanti-ligand, conjugated anti-ligand-specific antibody fragments,conjugated biotin-avidin, and the like. Preferably theanti-ligand-specific antibodies, antibody fragments, or biotin-avidinare conjugated to fluorescent markers such as fluorescein or rhodamine,or to enzymes which catalyze the formation of products that can bequantified by their color or absorbance in the visible range, such as,β-galactosidase, alkaline phosphatase, or horseradish peroxidase.

In accordance with the present invention, the amount of anti-ligandbound to the adherent protein is then compared to a control sample orseries of control samples, in order to determine the absolute amount ofadherent protein present in the unknown sample. Any kind of controlsample may be used, so long as it contains known amounts of a substancewhich can be empirically related to the adherent protein of interest.Preferably, the control samples will contain known amounts of theadherent protein of interest. More preferably, the control samples willcontain known amounts of said adherent protein in purified orsubstantially purified form. Most preferably, the control samples willcontain known amounts of the purified adherent protein along with asufficient quantity of unrelated proteins to adjust the total proteinconcentration of the control sample to be about the same as that of theunknown samples.

The unrelated proteins are preferably proteins which do not cross-reactimmunologically with the adherent protein of interest. It is alsopreferred that the unrelated protein(s) aid in the binding of theadherent protein to the support medium in a manner analogous to which itwould bind from the biological fluid of interest. Unrelated proteins foruse in the practice of the present invention include proteins found inbiological fluids and bacterial and viral protein present duringinfections and other pathophysiological states.

When globular serum or plasma proteins are being assayed in accordancewith the present invention, one or more different globular or serumproteins are used as the unrelated protein or proteins. More preferably,the unrelated proteins are albumin (except in the case wherein theadherent protein being measured is albumin) and immunoglobulin G. Mostpreferably, the unrelated proteins are albumin, again except in the casewherein the adherent protein being measured is albumin, andimmunoglobulin G, both of the same taxonomic species as the adherentprotein of interest, e.g., if the adherent protein is from humans, theunrelated proteins are most preferably from humans.

With respect to lipophilic proteins in particular, the methodology ofthe present invention differs from previously described assays in thatthe noncovalent interactions of lipophilic proteins with other plasmaconstituents are disrupted, permitting the direct coating of microtiterwells with a fraction of the lipophilic protein which is proportional toits concentration. In accordance with the present invention, lipophilicprotein ligands are bound to microtiter plates in high salt at elevatedtemperature. Another feature of the present invention is the use ofpurified lipoprotein standard enriched in the ligand of interest andnormalized to the total protein concentration of the samples. Thismethodology enables the simple, accurate and reproducible detection andmeasurement of lipophilic serum or plasma proteins in clinical sampleson a large scale.

Although the present invention is useful in the assay of adherentproteins in general, it is especially useful for the assay of lipophilicproteins because it overcomes problems associated with previouslyavailable assays which make prior assays unsuitable for clinicalmeasurement of lipophilic proteins. One embodiment of the presentinvention described herein includes immunoassay procedures for SAA andApoAl that can be applied to serial monitoring of patients during largeclinical trials. The features of these assays are (1) standard referencecurves are constructed by addition of purified SAA-rich or ApoAl-richlipoprotein to a solution of human IgG and albumin at concentrationscorresponding to the test samples; (2) SAA or ApoAl ligands are coatedon microtiter wells in high salt at elevated temperature; and (3) serialdilutions are assayed simultaneously to insure that values for SAA orApoAl concentration are obtained from dilutions in the linear range ofthe standard curve.

The assays of the present invention do not require a delipidation step,since the lipophilic protein of interest is coated onto the supportmedium, e.g., microtiter wells, at high temperature and high salt.Elimination of the delipidation step is advantageous, becausedelipidation of samples is labor-intensive and time-consuming. Moreover,delipidation can be a source of error in the level of lipophilic proteinactually measured, since the process can degrade the protein or alterthe conformation of antigenic sites.

Furthermore, the present invention offers significant advantages overknown assays for lipophilic serum proteins in terms of sensitivity. Forexample, the concentration of SAA in patients with chronic conditionssuch as rheumatoid arthritis is lower than in experimental animalsundergoing acute inflammation. (See, e.g., Zuckerman and Suprenant,supra). The present invention allows accurate and reproduciblemeasurement of the low levels of SAA found in these patients, as well asallowing comparison of samples collected over a period of time andjuxtaposition of results.

The assays of the present invention also use significantly less anti-SAAantiserum than do known procedures, e.g., Dubois and Malmendier (op.cit., 1988), since affinity purification of the antibodies is notrequired. Similarly, the present invention uses less antibody than theprocedure of Saile et al., Clin. Chem. 34, 1988, which also employsaffinity purified antibodies for capture and detection of human SAA inaddition to requiring a sample delipidation step.

Examples 1 and 2 below illustrate the method of the present inventionwith respect to SAA. Example 3 illustrates the measurement of ApoA andApoB. In Examples 4 through 6, the standard curves prepared usingcontrol samples, clearly demonstrate the usefulness of the methods ofthe present invention in assaying adherent proteins.

By means of specific anti-ligands and appropriate standards severaladherent proteins of interest could be quantified in a single sampledilution distributed to numerous wells and samples in an individual wellcould be analyzed by using separable detection systems.

The invention will be further understood with reference to the followingexamples, which are purely exemplary in nature, and not meant to beutilized to limit the scope of the invention.

EXAMPLE 1 Preparation of Standard

SAA and ApoAl were isolated as HDL complex by ultracentrifugation from apool of SAA-rich serum obtained from patients with gout and acutepneumonia (Roseff et al., 1987). After centrifugation at 2000×g, 4° C.,for 15 minutes, solid KBr (reagent grade, FisherScientific, Medford,Mass.) was added to adjust the plasma to a density of 1.21 gm/cc.Aqueous KBr solution, density 1.21 g/cc, was overlaid on up to 5 ml ofadjusted plasma. The tubes were centrifuged in a SW41 rotor for 36 hrs.at 15° C. Seven equal fractions were removed sequentially from the tubesby aspiration. The fractions were dialyzed against phosphate bufferedsaline (PBS) and stored at 4° C. or -70°. Repeated freeze-thawing wasavoided.

The protein content of the fractions was determined using the BioRad(Richmond, Calif.) protein determination kit with crystalline bovineserum albumin (Calbiochem, San Diego, Calif.) as standard. The purityand physical characteristics of the SAA thus obtained was analyzed bypolyacrylamide gel electrophoresis as described below (see FIG. 1). Ithas consistently been found that more than 80% of total plasma SAA isrecovered with HDL following a single ultracentrifugation in KBr (FIG.1).

FIG. 1(a) is the SDS-polyacrylamide gel electrophoretic analysis ofSAA-rich plasma (1000 μg/ml) after ultracentrifugation of 5 ml of plasmain KBr, density 1.21 gm/cc for 40 hours at 15° C. Lanes 1 through 7contain 100 ug each of protein. These results show that between 80 and90% of total plasma SAA protein was recovered in the top three fractionsafter ultracentrifugation.

FIG. 1(b) is the comparison of SDS-polyacrylamide gel electrophoreticanalysis of the top three fractions of a plasma pool from 10 healthysubjects (SAA, 4 μg/ml) with the top fraction of SAA-rich plasma. Lanes1-3 contain 30 μg each of top fractions from normal plasma; Lane 4contains 30 μg of top fraction of SAA rich plasma. This gel shows thatno proteins of size similar to SAA were detected when plasma fromhealthy subjects was fractionated by the ultracentrifugation proceduredescribed herein.

The ApoAl content of the HDL standard fraction was determined in thesame manner. Densitometric scanning showed that the HDL standardcontained 9% ApoAl.

SDS Polyacrylamide Gel Electrophoresis

Presample buffer was prepared by mixing 1 ml of glycerol, 2.3 ml 10%SDS, 1.25 ml 0.5M Tris, pH 6.8, and 4.95 ml water. Loading buffer wasprepared by mixing 930 μl of presample buffer, 50 μl ofbeta-mercaptoethanol and 52 μl of 0.1% bromophenol blue.

HDL and plasma fractions were denatured by boiling for 5 minutes inloading buffer and the constituents were size fractionated on 11.4%polyacrylamide gels with a 5% stacking gel, both containing 6.4M urea.Two sizes of gel have been utilized; the larger is 16×18 cm, 1.5 mmthick with 100 μg protein per lane and the smaller is 7.3×10.2 cm, 0.75mm thick with 30 μg protein per lane. The larger gels were run at 250volts with tap water cooling for 3 to 6 hours, the smaller at 75 voltswith air cooling for 1.5-2 hours.

Gel bands of SAA were excised and the protein was eluted from the gel inthe Schleicher and Schuell (VWR, Medford, Mass.) electroelutionapparatus. The buffer employed was 0.025M Tris base, 0.19M glycine, 0.1%sodium dodecyl sulfate (SDS).

The percentage of SAA in the SAA/HDL fraction was determineddensitometrically by scanning of individual lanes of Coomassie stainedgels using the Electrophoresis Data Center (Helena Laboratories,Beaumont, Tex.). The relative SAA concentration was confirmed by ELISAanalysis.

FIG. 1(c) is the SDS-polyacrylamide gel electrophoretic analysis ofSAA/HDL standards by scanning of Coomassie blue stained gels. Lane 1:100 ug SAA/HDL containing 37 ug SAA; Lane 2: 100 ug SAA/HDL containing32 ug SAA by ELISA; Lane 3: 100 ug SAA/HDL containing 41 ug SAA byELISA; Lane 4: 100 ug SAA/HDL containing 9 ug SAA by ELISA. Fraction 3(FIG. 1C, Lane 4) was selected for use as a standard in subsequent ELISAexperiments; it contained 9% SAA protein (Table I).

FIG. 1(d) is the SDS-polyacrylamide gel electrophoretic analysis of SAAelectroeluted from Coomassie blue stained gels. Lane 1: 5 ug each ofmolecular weight markers: in descending order, ovalalbumin (43,000),alpha-chymotrypsinogen (25,700), betalactoglobin (18,400), lysozyme(14,300), bovine trypsin inhibitor (6,200) and insulin (3,000). Lane 2:Approximately 10 ug of SAA protein electroeluted from SAA bands asdepicted in FIG. 1(a). When SAA was recovered from gel slices byelectroelution, Coomassie blue remained tightly bound to the SAAmonomer, which migrated as expected slightly ahead of the 14 kdmolecular weight marker.

EXAMPLE 2 Measurement of SAA Preparation of Antibodies to SAA

Electroeluted SAA at concentration of 200 ug/ml was emulsified withcomplete Freund's adjuvant and injected into rats at multiple sites(Wood et. at., 1982). The rats were boosted at 3 week intervals, andserum was tested for an increase in antibody activity relative topreimmune serum by ELISA. The presence of SAA-specific antibodies weredetermined by the direct binding ELISA method of Zuckerman andSurprenant (1986).

The electroeluted SAA elicited antibodies at titers ranging from 1:100to 1:10,000 within 3 weeks of immunization. The titers remained elevatedfor at least 12 weeks. It was necessary to absorb serum obtained laterin the immunization regimen with normal human serum or plasma. Afterabsorption with plasma lacking detectable SAA (DeBeer et al., 1982), theantibodies reacted specifically with SAA-rich HDL, and did not reactwith HDL from normal plasma.

Rabbit antibodies were raised to human AA proteins as previouslydescribed (Linke et al., 1975). Rabbit antibodies to human SAA werepurchased from Calbiochem (San Diego, Calif.) and a reference sample waskindly provided by Drs. A. Strachan and F. DeBeer (University ofStellenbosch, Tygerberg, South Africa).

ELISA of SAA

Anticoagulated plasma was stored frozen at -85° C. until analysis. Arepresentative assay consisted of 4 microtiter plates, each containingthe same 9 concentrations of SAA/HDL in triplicate wells. Plasma sampleswere diluted 1:10 by adding 10 μl of plasma to 90 μl of phosphatebuffered saline (0.10M sodium phosphate, 0.15M sodium chloride. pH 7.2(PBS) in the wells of flexible polyvinyl chloride microtitration plates.Fifteen μl aliquots were transferred to wells containing 135 μl of 3MKBr in 0.1M sodium bicarbonate, pH 9.6 (1:100) dilution. Immediatelyafter mixing, 50 μl aliquots were transferred to another platecontaining 100 μl of 3M KBr solution (1:300). Three additionalthree-fold dilutions (1:900, 1:2700 and 1:8100) encompass a linear rangefor SAA concentrations of up to 1000 μg/ml. Fifty μl was discarded fromthe final plate.

Standard SAA/HDL preparation was serially diluted in a correspondingdilution (1:100, 1:300, etc.) of human IgG (Calbiochem, San Diego,Calif.) 13 mg/ml and albumin (Calbiochem, San Diego, Calif.) 45 mg/ml,stock concentrations. Six μl of SAA 1.4 mg/ml, 9% SAA was added to 200μl of diluted IgG/albumin solution and after mixing, 100 μl wastransferred to 100 μl of solution in 8 successive dilutions.

The wells of the microtiter plates were coated with SAA by overnightincubation at 60° C. The next day, the wells were emptied andnonspecific sites were blocked by addition of a 5% solution of dry milkin 0.02M phosphate buffer containing 0.05% Tween-20, pH 7.4, andincubation for 1 hour at room temperature. The plates were then washedthree times with rinse buffer (0.02M phosphate, 0.05% Tween-20, pH 7.4).

The wells were incubated with 100 μl of a dilution of rat or rabbitpolyclonal antiserum (1:300 or greater dilution) in rinse buffer for 90minutes at 37° C. The plate was rinsed three times with rinse buffer,and 100 μl of a 1:1000 dilution of peroxidase conjugated goat anti-rator anti-rabbit IgG (Calbiochem) was added to each well. Incubation wascarried out for 90 minutes at 37° C.

The plates were then washed nine times with rinse buffer. Substrate wasprepared by dissolving 5.5 mg o-phenylenediamine dihydrochloride (PDA)in 10 ml citrate buffer (0.1M sodium citrate, 0.1M disodium monohydrogenphosphate pH 5). Immediately before addition to the microtiter wells, 33μl of 3% hydrogen peroxide was added to the PDA solution. Aliquots of100 ul of substrate solution were added to each well, and colordevelopment was monitored in the VMax Automated ELISA reader (MolecularDevices, San Diego, Calif.) at 450 nm. After about 5 minutes, 100 ul of1M sulfuric acid was added and the plates were read at 490 nm. Data wereanalyzed by linear regression analysis of the standard curve obtained byplotting the log of ng/well SAA vs the absorbance at 490 nm. Thecoefficient of correlation of the points in the linear range of thecurve was routinely >0.99. The average within plate coefficient ofvariation was less than 5%; the average between plate coefficient ofvariation was less than 20%.

Seven control samples and 16 samples comprised a single run, which wasrepeated to confirm values. This, together with the control valuesprovided assurance that concentration values have been determined fromthe most appropriate dilutions (Table III). A value was always obtainedfrom plate 1, 1:100 dilution. If the SAA value in ng/well was twice orgreater the blank value for plate b, or thrice the value for plates cand d, the blank was subtracted from the gross ng/well and the net valuewas multiplied by the dilution factor to give the final SAAconcentration in ug/ml for that plate.

It is desirable that the protein concentration of the standard curve beas similar as possible to the samples being tested if absolute, ratherthan relative, concentration values are needed. Standard curves for theSAA assay were linear over an approximately 20 fold range (FIG. 4). InFIG. 4, SAA/HDL in the amounts indicated was added to triplicate wellsof plates a through d containing: a-1:100, b-1:300, c-1:900, d-1:2700dilution human IgG (13 mg/ml and albumin (45 mg/ml).

The sensitivity of the assay increased as the amount of competing plasmaprotein decreased, to <10 ng/well at a 1:2700 dilution of plasma. Thehighest concentration of plasma that was routinely analyzed was a 1:100dilution, and the minimum concentration of plasma SAA that could bedetected by the method of the present invention was approximately 1μg/ml.

The blank value, due to cross-reactivity of goat anti-rabbit conjugatewith the human IgG, varied between 5 and 25 ng/well, according toefficacy of absorption of the conjugate serum with normal human plasmaand with IgG and albumin. This value, generally on the order of 10 μg/mlis subtracted from each sample concentration value (Table III). Thecoefficients of correlation of the standard curves are routinely≧0.99.

Comparable results were obtained with commercial rabbit anti-SAA, rabbitanti-AA antiserum and rat anti-AA.

Effect of Temperature on SAA Binding

Heat was found to promote proportional and reproducible coating of SAAto wells (FIGS. 2 and 3). SAA/HDL standard was serially diluted inbicarbonate solution, pH 9.6 to give the amounts of SAA per well shownin FIG. 2. The plates were sealed and incubated at 4° C. --O-- or 60° C.--O--. The amount of SAA bound by the wells was proportional to theabsorbance at 490 nm. The average coefficient of variation of triplicatewells was less than 5 percent.

FIG. 2 shows that coating the wells with the SAA/HDL standard proteinovernight at 60° C. in bicarbonate solution pH 9.6 enhanced bindingand/or exposure of antigenic determinants as compared with overnightbinding at 4° C. Direct binding of SAA from serum and plasma samples wasalso increased at elevated temperatures (Table II). However, the amountof SAA bound to wells from serum or plasma containing a greater amountof SAA was much less than from isolated HDL fractions, indicating asubstantial dampening effect of serum components upon binding.

Effect of salt on SAA Binding

Salt was also found to promote proportional and reproducible coating ofSAA to wells. FIG. 3 shows the inhibitory effect of albumin on SAAbinding to wells of microtiter plates and its reversal by heating in thepresence of salt. In FIG. 3(a), a mixture of SAA/HDL standard (1200ng/ml) and albumin (10 μg/ml) were serially diluted in bicarbonatesolution, pH 9.6, and wells were coated by overnight incubation at 4° C.--O--. SAA/HDL standard only was incubated overnight at 4° C. --O--.

In FIG. 3(b), SAA/HDL with and without albumin was diluted as describedfor FIG. 3(a), but coating was carried out by incubation at 60° C. in 3MKBR overnight.

Binding of SAA to wells was inhibited in the presence of albumin,particularly at 4° C. (FIG. 3a). Greater binding of SAA from HDLfractions and from plasma was observed when plasma was diluted in 3M KBrand binding was carried out at 60° C. Salt and elevated temperature wereobserved to diminish the inhibitory effect of albumin on SAA binding(FIG. 3B).

EXAMPLE 3 Measurement of ApoAl and ApoB

A. ApoAl

Anticoagulated plasma was stored frozen at -85° C. until analysis. Arepresentative assay consisted of 4 microtiter plates, each platecontaining the same sample concentration in triplicate wells. Plasmasamples were diluted 1:20 by adding 10 μl of plasma to 190 μl of PBS inthe wells of microtitration plates. Ten μl of the first dilution wasfurther diluted into 190 μl PBS, to yield a 1:400 dilution of theoriginal sample. Ten μl aliquots of the 1:400 dilution was transferredto triplicate wells containing 190 μl of 3M KBr in 0.1M sodiumbicarbonate, pH 9.6 (1:8000) dilution. Immediately after mixing, 100 μlaliquots were transferred to another plate containing 100 μl of 3M KBrsolution (1:16,000). Two additional two-fold dilutions (1:32,000 and1:64,000) encompass a linear range for ApoAl concentrations of up to 7mg/ml. One hundred μl was discarded from the final plate.

Standard ApoAl/HDL preparation was serially diluted in a correspondingdilution (1:8000, 1:16,000, etc.) of human IgG (Calbiochem, San Diego,Calif.) 13 mg/ml and albumin (Calbiochem, San Diego, Calif.) 45 mg/ml,stock concentrations. 5.2 μl of HDL containing ApoAl 1.5 mg/ml, 9% ApoAlwas added to 200 μl of diluted IgG/albumin solution and after mixing,100 μl was transferred to 100 μl of solution in 11 successive dilutions.

The wells of the microtiter plates were coated with ApoAl by overnightincubation at 60° C. The next day, the wells were emptied andnonspecific binding sites are blocked with a 5% solution of dry milk in0.02M phosphate buffer containing 0.05% Tween 20, pH 7.4, by incubatingfor 1 hour at room temperature. The plate was washed three times withrinse buffer without dry milk.

The wells were incubated with 100 μl of a dilution of rabbit polyclonalanti-ApoAl antiserum (Calbiochem, LaJolla, Calif.) (1:3000 dilution) inrinse buffer for 90 minutes at 37° C. The plate was rinsed three timeswith rinse buffer, and 100 μl of a 1:1000 dilution of peroxidaseconjugated goat anti-rabbit IgG was added to each well. Incubation wascarried out for 90 minutes at 37° C.

The plates were then washed nine times with rinse buffer. Substrate wasprepared by dissolving 5.5 mg o-phenylenediamine dihydrochloride (PDA)in 10 ml citrate buffer (0.1M sodium citrate, 0.1M disodium monohydrogenphosphate pH 5). Immediately before addition to the microtiter wells, 33μl of 3% hydrogen peroxide was added to the PDA solution. Aliquots of100 μl of substrate solution were added to each well, and colordevelopment was monitored in the VMax Automated ELISA reader (MolecularDevices, San Diego, Calif.) at 450 nm. After about 5 minutes, 100 μl of1M sulfuric acid was added and the plates were read at 490 nm. Data wereanalyzed by linear regression analysis of the standard curve obtained byplotting the log of ng/well SAA vs the absorbance at 490 nm. Thecoefficient of correlation of the points in the linear range of thecurve was routinely>0.99. The average within plate coefficient ofvariation was less than 5%; the average between plate coefficient ofvariation was less than 20%.

Results of the ApoAl assay are shown in Table IV. FIG. 6 is a standardcurve for the ApoAl ELISA of the present invention.

FIG. 7 shows a comparison of the ELISA of the present invention with aconventional immunoturbidimetric assay for ApoAl. Theimmunoturbidimetric assay was performed using a kit from SigmaDiagnostics (St. Louis, Mo.).

B. ApoB

In the ApoB assay, the procedure was as described in Example 3.A. abovefor the standard ApoAl/HDL preparation, except that an LDL preparationfrom Calbiochem was used as standard. Primary and secondary antibodyconcentrations were 1:1000 and 1:5000.

FIG. 8 shows a standard curve for the ApoB ELISA of the presentinvention.

EXAMPLE 4 Measurement of Cytokines: IL-1 beta and TNF alpha

In the measurement of the cytokines, IL-1 beta and TNF alpha, procedureswere as described for the standard apoAI/HDL preparation in Example 3,above except that purified recombinant generated cytokines were used andthat IgG/albumin was not used in the coating buffer except as indicatedfor IL-1, where it was used at 1:100 of the stock concentrations. Thedilutions for the primary and secondary antibodies were 1:1000.

FIG. 9 shows standard curves for control samples containing IL-1 beta inthe presence and absence of IgG/albumin. FIG. 10 shows a standard curvefor TNF-alpha.

EXAMPLE 5 Measurement of CRP

In the following assay, primary and secondary antibody concentrationswere 1:1000 to 1:5000.

Purified CRP was purchased from Sigma and rabbit antibodies to purifiedCRP from Calbiochem.

FIG. 11 shows standard curves obtained for control samples containingpurified human CRP in the presence (FIG. 11A) and absence (FIG. 11B) ofpurified human IgG and albumin. Using the standard curve with albuminand IgG, clinically reasonable values were obtained i.e. CRP was notdetectable in normal plasma and was 11.8 ug/ml in the plasma of arheumatoid arthritis patient. The plasma samples were diluted 1:100.

EXAMPLE 6 Measurement of Albumin

In the following assay, primary and secondary antibody concentrationswere 1:1000 and 1:5000.

Purified human albumin and rabbit antibodies to albumin were purchasedfrom Calbiochem.

FIG. 12 shows standard curves obtained for control samples containingpurified serum albumin in the presence (FIG. 12A) and absence (FIG. 12B)of purified human IgG.

Table V shows values obtained by a preliminary ELISA for albuminconcentrations in plasma from a healthy individual (NHP) and arheumatoid arthritis patent (RA). In order to optimize this ELISA, it isdesirable to add other unrelated proteins, such as lipoprotein, inaddition to IgG to the standard curve in order to obtain absolute valuesfor albumin, because values obtained in the presence of IgG were lowerthan in the absence of IgG. The plasma samples were diluted 1:40.000.

                  TABLE I                                                         ______________________________________                                        Comparison of SAA content determined by gel scanning and                      by ELISA                                                                      SAA/HDL                                                                       (ng analyzed)         % SAA                                                   ______________________________________                                                              ELISA                                                   700                   8,12, 9                                                 350                   6,14, 8,10                                              175                   5,13, 8, 9                                               88                   4,14,12, 7                                              Average SAA content   9.3 ± 3.1%                                                                 Gel Scan                                                100                   9.3                                                     ______________________________________                                    

SAA content of lane 4, FIG. 2B, was compared by ELISA with SAA/HDLstandard, 37% SAA. Coating of wells was carried out at 4° C. inbicarbonate buffer, pH 9.3, lacking KBr. Gel scanning of Coomassiestained gel was carried out as described in the Methods Section.

                  TABLE II                                                        ______________________________________                                        Effect of temperature on SAA binding from plasma samples.                     A 490                                                                         SAA concentration  4° C.                                                                         60° C.                                       ______________________________________                                          4 ug/ml          0.18   0.24                                                 32                0.15   0.28                                                 112               0.27   0.36                                                 585               0.55   0.42                                                1000               0.59   0.68                                                ______________________________________                                    

Plasma samples were diluted 1:10 in PBS and then 1:10 in KBr bicarbonatebuffer, pH 9.6. Incubation was carried out overnight at indicatedtemperature, and the amount of SAA bound was quantified by measurementof absorbance at 490 nm after blocking and incubation with antibodies asdescribed in the Methods Section.

                                      TABLE III                                   __________________________________________________________________________    SAA Direct Binding ELISA Data and Calculations                                SAA (ng/well)                                                                 Run 1             Run 2         SAA                                           Sample                                                                            a   b  c   d  a   b  c   d  (ug/ml)                                       __________________________________________________________________________     1  17  10 8   4  19  9  6   3  6,9                                            2  17  8  6   3  16  8  5   3  6,6                                            3  16  8  6   3  16  8  5   3  5,6                                            4  15  8  6   3  15  8  5   3  4,5                                            5  15  8  6   3  14  8  5   3  3,4                                            6  16  7  6   3  14  8  5   3  5,4                                            7  16  8  6   3  14  8  6   3  5,4                                            8  28  14 9   4  28  13 7   4  17,24,18,21                                    9  31  15 10  4  42  17 9   4  20,27,32,33                                   10  20  10 9   4  25  11 7   4   9,15                                         11  14  7  6   3  14  8  5   3  3,4                                           12  14  8  6   3  16  8  6   3  3,6                                           13  48  29 15  6  85  35 16  7  37,69,99,104                                                                  75,87,108                                     14  14  9  7   4  16  9  6   3  3,6                                           15  15  8  7   4  15  9  6   3  4,5                                           16  14  8  6   3  16  8  6   3  3,6                                           17  44  17 10  4  41  16 7   4  33,33,31,30                                   18  168 50 20  8  117 37 12  5  157,132,144                                                                   107,93                                        19  112 32 17  6  57  20 9   5  101,78,117                                                                    47,42,45                                      20  99  58 37  20 143 68 35  20 294,486                                                                       279,459                                       21  145 123                                                                              61  31 194 131                                                                              53  31 513,783                                                                       441,756                                       __________________________________________________________________________

                  TABLE IV                                                        ______________________________________                                        Comparison of ApoAl Direct Binding ELISA and                                  Turbidimetric Assay                                                           Sample        Kit (mg/ml)                                                                              ELISA (mg/ml)                                        ______________________________________                                        1. NHP        1.74       2.50                                                 2. MED        0.76       0.75                                                 3. APP        1.08       0.64                                                 4. P1         1.00       0.97                                                 5. P2         1.36       1.50                                                 6. Kit Control I                                                                            0.97       1.04                                                 7. Kit Control II                                                                           1.50       1.56                                                 ______________________________________                                    

                  TABLE V                                                         ______________________________________                                        Albumin Direct Binding ELISA: Normal Human Plasma and                         Plasma From Rheumatoid Arthritis Patient                                      Sample in mg/ml                                                                          NHP      RA 1578  IgG                                              ______________________________________                                        Curve w/o IgG                                                                              8.65       10.64                                                 Curve w/IgG  5.73       7.22     0.05                                         ______________________________________                                    

It is understood that the examples and embodiments described herein arefor illustrative purposes only, and that various modifications orchanges in light thereof that will be suggested to persons skilled inthe art are to be included in the spirit and purview of this applicationand the scope of the approved claims.

What is claimed is:
 1. A method of determining the amount of an adherentprotein in a sample, the adherent protein being capable of forming anon-specific hydrophobic interaction with a support medium, the methodcomprising:a. contacting the sample at high salt concentration whereinthe salt comprises KBr at a concentration from about 0.15M to aboutsaturation and elevated temperature from about 40° C. to about 65° C.with a support medium having an affinity for the adherent protein underconditions to effect binding of the adherent protein to the medium; b.contacting the support medium obtained in step (a) with at least oneanti-ligand for the adherent protein under conditions to promotespecific binding of the anti-ligand to the adherent protein; c.detecting and measuring the amount of anti-ligand bound to the adherentprotein; and d. either (i) relating the amount of bound anti-liganddetermined in step (c) with the amount of bound anti-ligand measured forat least one control sample prepared in accordance with steps (a) to(c), said control sample comprising one or more unrelated proteins knownto be free of the adherent protein, or (ii) relating the amount of boundanti-ligand measured for samples containing known amounts of adherentprotein, said samples comprising purified adherent protein in a solutionof one or more unrelated proteins, and prepared in accordance with steps(a) to (c).
 2. The method of claim 1, wherein the adherent protein is alipophilic serum protein, a cytokine or a globular serum or plasmaprotein or a pentraxin.
 3. The method of claim 2, wherein the lipophilicserum protein is one associated with HDL, LDL, or VLDL.
 4. The method ofclaim 3, wherein the lipophilic serum protein is one associated withHDL.
 5. The method of claim 2, wherein the lipophilic serum protein isserum amyloid A, apoprotein Al, apoprotein B, tumor necrosis factor, orinterleukin-6.
 6. The method of claim 1, wherein the pH of step (a) isfrom about pH 8 to about pH
 11. 7. The method of claim 1, wherein theunrelated protein is a globular serum protein.
 8. The method of claim 1,wherein the unrelated proteins are immunoglobulin G and serum albumin.9. The method of claim 1, wherein the unrelated protein is serumalbumin.
 10. The method of claim 1, wherein the support medium comprisesa solid medium.
 11. The method of claim 10, wherein the solid medium ismicrotiter plates, plastic beads, resins, or magnetic beads.
 12. Themethod of claim 1, wherein the anti-ligand is a polyclonal antiserum, amonoclonal immunoglobulin, a receptor molecule, or a lipid transportmolecule.
 13. The method of claim 1, wherein the anti-ligand comprisesan anti-ligand-specific antibody, an anti-ligand-specific antibodyfragment, or biotin-avidin conjugated to a label.
 14. The method ofclaim 13, wherein the label is a fluorescent label.
 15. The method ofclaim 14, wherein the fluorescent label is fluorescein or rhodamine. 16.The method of claim 13, wherein the label is an enzyme.
 17. The methodof claim 16, wherein the enzyme is β-galactosidase, alkalinephosphatase, or horseradish peroxidase.
 18. A method of determining thepresence of an adherent protein in a sample, the adherent protein beingcapable of forming a non-specific hydrophobic interaction with a supportmedium, the method comprising:a. contacting the sample at high saltconcentration wherein the salt comprises KBr at a concentration fromabout 0.15M to about saturation and elevated temperature from about 40°C. to about 65° C. with a support medium having an affinity for theadherent protein under conditions to effect binding of the adherentprotein to the support medium; b. contacting the support medium obtainedin step (a) with at least one anti-ligand for the adherent protein underconditions to promote specific binding of the anti-ligand to theadherent protein; and c. detecting anti-ligand bound to the adherentprotein.